Breast cancer targeting nanoparticle as well as preparation method and application thereof

Abstract

The embodiment of the invention provides breast cancer targeting nanoparticles as well as a preparation method and application thereof. According to the embodiment of the invention, the breast cancer targeting nanoparticles are synthesized by preparing the pH sensitive polymer, carrying out co-coupling modification on the pH response polymer and the folic acid albumin nano-carrier and utilizing a controlled desolvation method; the breast cancer targeting nanoparticle is combined with a nano preparation passive targeting strategy, a tumor microenvironment stimulation response strategy and a folic acid active targeting strategy, the effects of an EPR effect, PEG shedding, charge overturning, folic acid ligand-receptor combination and the like are utilized, precise targeting and specific response of tumors are achieved, and targeted accumulation of drugs in a tumor microenvironment is comprehensively improved. Therefore, the breast cancer targeting nanoparticles prepared by the embodiment have the advantages of high targeting property, drug controlled release, improvement of solubility and absorptivity of indissolvable drugs and the like, so that the curative effect of the drugs can be enhanced, the toxic and side effects can be reduced, and the breast cancer targeting nanoparticles have wide application prospects in the field of anti-tumor treatment.

Description

Technical Field

[0001] The present application relates to the technical field of pharmaceutical preparations, and particularly relates to a breast cancer-targeted nanoparticle and its preparation method and use. Background Art

[0002] Triple-negative breast cancer (TNBC) refers to a subtype of breast cancer in which estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are negative. TNBC is highly invasive, has a high histological grade, extensive heterogeneity, and ductal histological features, and metastatic TNBC is associated with a low overall survival rate.

[0003] Currently, the treatment methods for TNBC clinically mainly include tumor resection, chemotherapy, radiotherapy, and immunotherapy, etc. Among them, chemotherapy is widely used, but there are still unsolved problems, such as the off-target effect causing toxic and side effects of chemotherapeutic drugs on normal tissues, the development of acquired drug resistance in tumor cells, etc.

[0004] In recent years, research on the preparation method and pharmacokinetics of albumin-based nanopharmaceuticals has shown that because of its functional advantages of prolonging the circulation time and efficiently targeting tumor lesions, it is beneficial to enhance the clinical treatment efficacy of drugs. However, there are still certain limitations in the preparation process and stability at present. For example: the preparation process is complex and cumbersome, the cost is high, which is not conducive to industrial large-scale production; the long-term stability of the preparation is insufficient, which may cause premature release of the drug during the in vivo circulation process, reducing the drug efficacy and increasing organ side effects.

[0005] Therefore, developing a targeted nanopreparation for breast cancer based on albumin nanomaterials is of great significance for improving the treatment methods of breast cancer. Summary of the Invention

[0006] The purpose of the present application is to provide a breast cancer-targeted nanoparticle and its preparation method and use.

[0007] To achieve the above purpose, the embodiments of the present application propose the following technical solutions:

[0008] In the first aspect, the embodiments of the present application provide a preparation method of a breast cancer-targeted nanoparticle, and the preparation method includes:

[0009] Dissolve folic acid in an organic solution, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and a catalyst, and react to obtain a folic acid activation solution; add the folic acid activation solution to an albumin solution and react to obtain Polymer I;

[0010] Dissolve methoxypolyethylene glycol - amine and 3,4,5,6 - tetrahydrophthalic anhydride in water, adjust the pH value to 8.0 - 8.5, and react to obtain an mPEGA - DCA solution; add 1 - ethyl - 3 - (3 - dimethylaminopropyl)carbodiimide hydrochloride and N - hydroxysuccinimide to the mPEGA - DCA solution, adjust the pH value to 7.4 - 8.0, and react to obtain Polymer II;

[0011] After mixing the aqueous solution of Polymer II and the aqueous solution of Polymer I, adjust the pH value to 7.4 - 8.0, and react to obtain Polymer III;

[0012] Dissolve Polymer III in water, adjust the pH value to 8.0 - 8.5 to obtain a Polymer III solution; dissolve BA in ethanol and add it to the Polymer III solution, and add ethanol during stirring to obtain an opalescent solution; the opalescent solution is stirred, centrifuged, and filtered to obtain breast cancer - targeting nanoparticles.

[0013] As an embodiment, the organic solvent is dimethyl sulfoxide.

[0014] As an embodiment, the catalyst is 4 - dimethylaminopyridine.

[0015] As an embodiment, the step of dissolving BA in ethanol and adding it to the Polymer III solution, and adding ethanol during stirring to obtain an opalescent solution includes:

[0016] Dissolve BA in ethanol and add it to the Polymer III solution, and add ethanol at a rate of 1.0 - 2.0 mL / min during stirring to obtain an opalescent solution.

[0017] As an embodiment, the preparation method of the albumin solution includes:

[0018] Dissolve albumin in water to obtain a mixture;

[0019] Add an alkali reagent to the mixture and adjust the pH value of the mixture to 8 - 10 to obtain an albumin solution.

[0020] As an embodiment, the albumin is at least one of human serum albumin, bovine serum albumin, and recombinant human serum albumin.

[0021] In a second aspect, an embodiment of the present application provides a breast cancer - targeting nanoparticle, which is prepared by the preparation method described in the first aspect.

[0022] In a third aspect, an embodiment of the present application provides the use of the breast cancer - targeting nanoparticle described in the second aspect in the preparation of a drug for preventing, alleviating, or treating breast cancer.

[0023] As an implementation manner, the drug is a drug for promoting the active uptake ability of breast cancer cells.

[0024] As an implementation manner, the drug is an injectable solution or a drug for oral administration.

[0025] The embodiments of the present application have at least the following beneficial effects:

[0026] In the embodiments of the present application, a pH-sensitive polymer (Polymer II) is prepared, and the pH-responsive polymer and folic acid albumin nanocarrier (Polymer I) are co-conjugated and modified. The baicalin pH-responsive nanoparticles (breast cancer-targeting nanoparticles) are synthesized by the controlled desolvation method; the breast cancer-targeting nanoparticles combine the passive targeting of the nanopreparation, the tumor microenvironment-stimulated response, and the folic acid active targeting strategy, and utilize the EPR effect, PEG shedding, charge inversion, folic acid ligand-receptor binding and other effects to achieve the precise targeting and specific response of the tumor, and comprehensively improve the targeted accumulation of the drug in the tumor microenvironment.

[0027] Therefore, the breast cancer-targeting nanoparticles of this embodiment can actively target breast cancer cells, regulate the stadmin / STAT3 signaling pathway, inhibit the polarization of the tumor-derived exosome-induced macrophage M2 pro-tumor phenotype, and achieve its inhibitory effect on breast cancer growth by improving the breast cancer microenvironment. It is indicated that the breast cancer-targeting nanoparticles have good application prospects in the preparation of drugs for treating TNBC.

[0028] The additional aspects and advantages of the present application will be partially given in the following description, and these will become obvious from the following description, or will be understood through the practice of the present application. Description of the Drawings

[0029] Figure 1 Schematic diagram of the synthesis route of the breast cancer-targeting nanoparticles in Example 1;

[0030] Figure 2 1H NMR spectrum of the nanoparticles in Example 2;

[0031] Figure 3 FT-IR spectra of FA, BSA and FA-BSA of the nanoparticles in Example 2;

[0032] Figure 4 FT-IR spectra of BA, mPEGA-DCA-NHS, BSA-FA@mPEG and BA-AN-FA@mPEG of the nanoparticles in Example 2;

[0033] Figure 5 TGA diagrams of BSA, FA-BSA, BA-BSANPs of the nanoparticles in Example 2;

[0034] Figure 6Thermogravimetric analysis diagrams of nanoparticles BA, BSA-FA@mPEG, and BA-AN-FA@mPEG in Example 2;

[0035] Figure 7 Schematic diagram of the appearance morphology of BA-AN-FA@mPEG aqueous solution in Example 2;

[0036] Figure 8 Transmission electron microscopy morphology schematic diagram of BA-AN-FA@mPEG in Example 2;

[0037] Figure 9 Particle size schematic diagram of BA-AN-FA@mPEG in Example 2;

[0038] Figure 10 Zeta potential schematic diagram of BA-AN-FA@mPEG in Example 2;

[0039] Figure 11 Schematic diagram of the encapsulation efficiency stability test results of nanoparticles BA-AN-FA@mPEG in Example 2;

[0040] Figure 12 Schematic diagram of the hydrolysis rate test results of BA-AN-FA@mPEG at different pH values in Example 2;

[0041] Figure 13 Schematic diagram of the particle size and potential changes of BA-AN-FA@mPEG at different pH values in Example 2;

[0042] Figure 14 Schematic diagram of the pH-sensitive drug release curves of BA-AN-FA@mPEG and BA in Example 2;

[0043] Figure 15 Schematic diagram of the qualitative uptake test results of MDA-MB-231 cells on BA, BA-AN-FA, and BA-AN-FA@MPEG at pH 7.4 in Example 3;

[0044] Figure 16 Schematic diagram of the qualitative uptake test results of MDA-MB-231 cells on BA, BA-AN-FA, and BA-AN-FA@MPEG at pH 6.5 in Example 3;

[0045] Figure 17 Schematic diagram of the quantitative uptake test results of MDA-MB-231 cells on C-6, BA-AN-FA, and BA-AN-FA@MPEG; the left side shows the test results at pH 7.4, and the right side shows the test results at pH 6.5;

[0046] Figure 18Schematic diagram of the activity of BA, BA-AN-FA@mPEG, and BSA-FA@mPEG on MDA-MB-231 cells in Example 3;

[0047] Figure 19 Cell cycle test chart of MDA-MB-231 in Example 3;

[0048] Figure 20 Cell cycle test chart of MDA-MB-231 in Example 3;

[0049] Figure 21 Cell cycle test chart of MDA-MB-231 in Example 3;

[0050] Figure 22 Flow cytometry chart of apoptosis of MDA-MB-231 in Example 3;

[0051] Figure 23 Flow cytometry chart of apoptosis of MDA-MB-231 in Example 3;

[0052] Figure 24 Flow cytometry chart of apoptosis of MDA-MB-231 in Example 3;

[0053] Figure 25 Schematic diagram of the detection results of the co-culture effect and mechanism study (before drug administration) in Example 3;

[0054] Figure 26 Schematic diagram of the detection results of the co-culture effect and mechanism study (before drug administration) in Example 3;

[0055] Figure 27 Schematic diagram of the detection results of the co-culture effect and mechanism study (before drug administration) in Example 3;

[0056] Figure 28 Schematic diagram of the detection results of the co-culture effect and mechanism study (before drug administration) in Example 3;

[0057] Figure 29 Schematic diagram of the detection results of the co-culture effect and mechanism study (after drug administration) in Example 3;

[0058] Figure 30 Schematic diagram of the detection results of the co-culture effect and mechanism study (after drug administration) in Example 3. Detailed implementation methods

[0059] Next, the technical solutions in the embodiments will be clearly and completely described in conjunction with the embodiments of the present application and the accompanying drawings. Obviously, the embodiments described below are only a part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts shall fall within the scope of protection of the present application.

[0060] It should be understood that when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, wholes, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components, and / or their combinations.

[0061] It should also be understood that the terms used in the specification of the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present application. As used in the specification of the embodiments of the present application and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" are intended to include the plural forms.

[0062] First, some terms and materials involved in this embodiment will be explained below to facilitate the understanding of those skilled in the art.

[0063] FA: Folic acid.

[0064] BA: Baicalin, and the structural formula of BA is

[0065] EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide.

[0066] NHS: N-Hydroxysuccinimide.

[0067] DMAP: 4-Dimethylaminopyridine.

[0068] DMSO: Dimethyl sulfoxide.

[0069] BSA: Bovine serum albumin.

[0070] mPEG-NH2: Or mPEGA, methoxypolyethylene glycol-amine.

[0071] DCA: 3,4,5,6-Tetrahydrophthalic anhydride.

[0072] EDC·HCl: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0073] BA-AN-FA@mPEG: Or pH-sensitive folic acid albumin nanocarrier, namely breast cancer targeting nanoparticles.

[0074] Unless otherwise specified, the water used in this example is ultrapure water.

[0075] Next, the breast cancer-targeted nanoparticles of this example, their preparation method and uses will be described in detail.

[0076] First, the preparation method of the breast cancer-targeted nanoparticles of the first aspect of this example will be described.

[0077] Preparation Method

[0078] This example provides a preparation method of breast cancer-targeted nanoparticles, and this preparation method includes:

[0079] (1) Dissolve folic acid in an organic solution, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and a catalyst, and react to obtain a folic acid activation solution; add the folic acid activation solution to an albumin solution and react to obtain Polymer I.

[0080] In step (1), by connecting folic acid molecules with N-hydroxysuccinimide (NHS), specific targeting and functions are achieved; at the same time, folic acid, as an important vitamin, has the ability to specifically bind to folate receptors on the cell surface. Based on this, folic acid-conjugated albumin nanocarriers can be used to deliver small molecule drugs, enter cells through the mediation of folate receptors, thereby achieving targeted delivery to tumor cells and reducing side effects.

[0081] (2) Dissolve methoxypolyethylene glycol-amine and 3,4,5,6-tetrahydrophthalic anhydride in water, adjust the pH value to 8.0 - 8.5, and react to obtain an mPEGA-DCA solution; add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the mPEGA-DCA solution, adjust the pH value to 7.4 - 8.0, and react to obtain Polymer II;

[0082] Step (2) is to obtain a pH-sensitive polymer (Polymer II) to provide reactants for the coupling reaction in the next step (step 3).

[0083] Among them, in step (2), mPEG-NH2 and DCA are used to react to generate mPEGA-DCA, and react with a condensing agent (EDC·HCl) and a carboxyl activating agent (NHS) to obtain Polymer II.

[0084] Specifically, the mPEG-NH2 selected in this step has amphiphilicity and good biocompatibility. Using mPEG-NH2 to modify the surface of the nanopreparation can increase the relative molecular mass and reduce renal elimination. At the same time, by virtue of its hydrophilic property, a hydration film can be formed on the surface of the nanopreparation, significantly improving the stability and water solubility of the drug, avoiding recognition by the reticuloendothelial system in the body, and prolonging the half-life of the drug.

[0085] (3) After mixing the aqueous solution of polymer II and the aqueous solution of polymer I, adjust the pH value to 7.4 - 8.0, and react to obtain polymer III.

[0086] In this step, the pH-responsive polymer and the folic acid-albumin nanocarrier (polymer I) are co-conjugated and modified. Specifically, through mPEG conjugation, the immunogenicity of biomolecules can be reduced, their stability against proteolytic enzymes can be increased, water solubility can be improved, and the half-life in vivo can be prolonged. At the same time, the therapeutic effect can be enhanced.

[0087] Among them, FA on polymer I can react with polymer II to form an amide bond under alkaline conditions, and this bond is easily broken under acidic conditions, causing the dissolution of the nanoparticle skeleton. The amide bond serves as a conjugate design linker for baicalin, which has acid-sensitive characteristics and can remain relatively stable at pH 7.4. This can enable the conjugate to remain stable before reaching the target and release the drug rapidly after reaching the lesion.

[0088] (4) Dissolve polymer III in water, adjust the pH value to 8.0 - 8.5 to obtain a polymer III solution; dissolve BA in ethanol and add it to the polymer III solution, and add ethanol during stirring to obtain an opalescent solution; after stirring, centrifuging, and filtering the opalescent solution, breast cancer-targeted nanoparticles are obtained.

[0089] In step (4), the baicalin pH-responsive nanoparticles (breast cancer-targeted nanoparticles) are synthesized by the controlled desolvation method; specifically, in this example, the dehydration effect of the organic solvent on albumin is utilized to cause the aggregation of albumin molecules and form nanoparticles at the nanoscale. At the same time, the drug (BA) is encapsulated in the albumin nanoparticles by covalent binding.

[0090] Therefore, this example is essentially to optimize the delivery of baicalin and develop a multifunctional composite baicalin-targeted drug delivery nanoparticle integrating passive targeting, active targeting, and stimulus-responsive mechanisms.

[0091] Based on this, the breast cancer-targeted nanoparticles prepared in this example combine the passive targeting of the nanopreparation, the tumor microenvironment stimulus response, and the folic acid active targeting strategy, and utilize the EPR effect, PEG shedding, charge flipping, folic acid ligand-receptor binding, etc. to achieve precise targeting and specific response of the tumor, comprehensively enhancing the targeted accumulation of the drug in the tumor microenvironment.

[0092] Therefore, the breast cancer-targeting nanoparticles prepared in this example have multiple advantages such as high targeting, controlled drug release, increased solubility and absorption rate of poorly soluble drugs, etc., which can enhance the efficacy of drugs and reduce toxic side effects, showing broad application prospects in the field of anti-tumor therapy.

[0093] The above preparation method will be further described below.

[0094] Among them, the organic solvent used in step (1) is dimethyl sulfoxide, and the catalyst is 4-dimethylaminopyridine.

[0095] Step (1), the preparation method of the albumin solution, includes:

[0096] Dissolve albumin in water to obtain a mixed solution;

[0097] Add an alkali reagent to the mixed solution to adjust the pH value of the mixed solution to 8-10 to obtain an albumin solution.

[0098] As an implementation manner, the albumin is at least one of human serum albumin, bovine serum albumin, and recombinant human serum albumin.

[0099] Preferably, the albumin is bovine serum albumin (BAS). In this example, BAS is used as an exemplary albumin for verification and description.

[0100] Among them, the above alkali reagent is preferably sodium hydroxide (NaOH); the pH value of the mixed solution is 8-10, preferably 9-10, such as 9.5, 9.8, 9.9, etc.

[0101] Generally, after adding the alkali reagent, an albumin solution is obtained by ultrasonic dissolution.

[0102] In this example, after the albumin solution is prepared, it is conjugated with folic acid. The folic acid-conjugated albumin nanocarrier can be used to deliver small molecule drugs, enter cells through the mediation of folic acid receptors, thereby achieving targeted delivery to tumor cells and reducing side effects.

[0103] Generally, in the above steps (1)-(4), stirring is carried out during the reaction process to accelerate the reaction or make the reaction more complete. In particular, step (1) is preferably carried out at room temperature in the dark.

[0104] In this example, unless otherwise specified, the relevant reaction processes of this example can be carried out at room temperature.

[0105] In step (4), BA is dissolved in ethanol and added to the polymer III solution, and ethanol is added during stirring to obtain an opalescent solution, including:

[0106] Dissolve BA in ethanol and add it to the polymer III solution. While stirring, add ethanol at a rate of 1.0 - 2.0 mL / min to obtain an opalescent solution.

[0107] Among them, by adding ethanol to the albumin solution containing BA, due to the dehydration effect of ethanol on albumin, albumin molecules aggregate to form nanoparticles at the nanoscale, and BA is encapsulated (coated) in the albumin nanoparticles.

[0108] Next, the synthesis process of breast cancer-targeted nanoparticles will be further clarified in combination with specific preparation methods.

[0109] Exemplarily, the preparation method of breast cancer-targeted nanoparticles includes:

[0110] (1) Preparation of Polymer I: Folate Albumin Nanocarrier (BSA-FA)

[0111] Weigh 13.3 mg of FA and add it to 5 mL of DMSO solution. Add 11.6 mg of EDC, 6.9 mg of NHS, and 3.0 mg of DMAP to the above reaction solution, and stir in the dark at room temperature for 12 h for activation. Weigh 40 mg of BSA and add it to 5 mL of water, and adjust the pH to 9.9 with 0.2 M NaOH solution, and dissolve it by ultrasonic treatment. Then, drop the FA activation solution into the BSA solution, and stir at 500 rpm / min in the dark at room temperature for 12 h. Dialyze with ultrapure water (14 KDa) for 72 h, and centrifuge at 12000 rpm for 10 min to obtain the folate albumin nanocarrier.

[0112] (2) Preparation of Polymer II: pH-Sensitive Polymer (mPEGA-DCA-NHS)

[0113] Stir and dissolve 100.0 mg of mPEG-NH2 (mPEGA) and 80.0 mg of 3,4,5,6-tetrahydrophthalic anhydride (DCA) in 5 mL of water, and adjust the pH to 8.0 - 8.5 with 0.2 M NaOH solution. After 12 h, centrifuge and filter (4000 rpm, 30 min), with a cut-off molecular weight of 1 KDa, to obtain the mPEGA-DCA polymer; freeze-dry the dialyzed mPEGA-DCA polymer; dissolve the freeze-dried mPEGA-DCA polymer in water to make a mPEGA-DCA solution with a concentration of 80 mg / mL;

[0114] 150.0 mg of EDC·HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 100.0 mg of NHS were added to the above mPEGA-DCA solution, and the mixture was stirred at room temperature for 12 h. Then, 0.2 M NaOH solution was added to adjust the pH to 7.4 - 8.0, followed by centrifugal filtration (4000 rpm, 20 min) with a cut-off of 1 KD. The purified polymer obtained was mPEGA-DCA-NHS.

[0115] (3) Polymer III: Preparation of pH-sensitive folic acid-conjugated albumin nanoparticles (BSA-FA@mPEG)

[0116] 10.0 mg of mPEGA-DCA-NHS was dissolved in 5 mL of water (pH 7.4), added to 20 mg of 5 mL BSA-FA aqueous solution, and 0.2 M NaOH solution was added to adjust the pH to 7.4. The mixture was stirred at 400 rpm / min at room temperature for 12 h. Unreacted polymers were removed by washing and centrifugation (12000 rpm, 10 min), and pH-sensitive folic acid-conjugated albumin nanoparticles were obtained by dialysis.

[0117] (4) Preparation of baicalin-loaded pH-sensitive folic acid-conjugated albumin nanoparticles (BA-AN-FA@mPEG)

[0118] 30 mg of BSA-FA@mPEG was placed in 3 mL of water, and 0.2 M NaOH solution was added to adjust the pH to 8.5. The mixture was stirred at room temperature for 10 min. 2.5 mg of BA was weighed and dissolved in 1 mL of absolute ethanol, and then added dropwise to the BSA-FA@mPEG solution. The mixture was stirred at 500 rpm / min for 30 min. 11 mL of ethanol was continuously added at a rate of 1.5 mL / min, and the solution changed from clear to a clear opalescent solution. The mixture was further stirred for 12 h. After centrifugation at 12000 rpm for 20 min, it was washed twice with ultrapure water and passed through a 0.45 μm organic filter membrane to obtain drug-loaded pH-sensitive folic acid-conjugated albumin nanoparticles.

[0119] Secondly, the breast cancer-targeted nanoparticles of the second aspect of this example will be described.

[0120] Breast cancer-targeted nanoparticles

[0121] Based on the preparation method of the first aspect, the breast cancer-targeted nanoparticles (or pH-sensitive folic acid-conjugated albumin nanoparticles) of this example have a pH-responsive polymer (Polymer II) and FA covalently conjugated to the albumin carrier, and baicalin is encapsulated. Thus, baicalin pH-responsive folic acid albumin nanoparticles (BA-AN-FA@mPEG) are prepared.

[0122] In the breast cancer-targeted nanoparticles of this embodiment, long-chain mPEG is present on the surface of albumin nanoparticles and has a charge inversion function. The ligand FA molecule can be hidden in the pH-responsive shell to avoid binding to normal cells without folate receptors or with folate receptors. Specifically, based on the EPR effect, BA-AN-FA@mPEG first accumulates passively in the acidic tumor microenvironment and responds to its weak acidic pH. The acid-sensitive amide bond connecting mPEG-NH2 to the albumin nanocarrier is hydrolyzed, and the long-chain mPEG falls off and degrades the acid-sensitive shell. Then, it is actively recognized by breast cancer cells overexpressing folate receptors, enters the cells to increase the specific drug accumulation amount and improve the bioavailability, thereby reducing the off-target effect at non-specific sites.

[0123] Therefore, based on the evaluation of the inhibitory effect of baicalin pH-responsive nanoparticles on breast cancer growth in this embodiment, the regulatory effect on macrophage phenotype will be clarified, and the regulatory mechanism of the tumor microenvironment will be explored and elaborated, which will provide empirical and scientific basis for the application of baicalin pH-responsive nanoparticles in the tumor microenvironment.

[0124] In summary, the breast cancer-targeted nanoparticles prepared in this embodiment have many advantages such as high targeting, controlled drug release, improving the solubility and absorption rate of poorly soluble drugs, etc., so as to enhance the efficacy of drugs and reduce the toxic and side effects, showing broad application prospects in the field of anti-tumor treatment.

[0125] Next, the use of the breast cancer-targeted nanoparticles in the third aspect of this embodiment will be described.

[0126] Use of Breast Cancer Targeted Nanoparticles

[0127] As mentioned above, the breast cancer-targeted nanoparticles prepared in this embodiment have many advantages such as high targeting, controlled drug release, improving the solubility and absorption rate of poorly soluble drugs, etc., so as to enhance the efficacy of drugs and reduce the toxic and side effects.

[0128] Based on this, the breast cancer-targeted nanoparticles provided in this embodiment can be used to prepare drugs for preventing, alleviating or treating breast cancer.

[0129] Specifically, the above-mentioned drugs are drugs for promoting the active uptake ability of breast cancer cells.

[0130] Exemplarily, the above-mentioned drugs are injectable solutions or drugs for oral administration.

[0131] Generally, when the above-mentioned breast cancer-targeted nanoparticles are made into corresponding drugs, the drugs also include pharmaceutically acceptable carriers and / or adjuvants.

[0132] The drug described in this embodiment has breast cancer-targeted nanoparticles as the active ingredient, and does not exclude changes in the formulation system and administration method, pharmaceutical salts obtained by simple chemical modification and adjustment of the above-mentioned breast cancer-targeted nanoparticles, and the combination of multiple compounds, etc.

[0133] For example, in this embodiment, one or more compounds in the breast cancer-targeted nanoparticles of this embodiment can be formulated as active ingredients in non-toxic, inert, and pharmaceutically acceptable carriers and / or adjuvants; the formulated drug can be administered through conventional routes, including but not limited to oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

[0134] For example, when the dosage form of the drug in this embodiment is a drug for oral administration, it contains a safe and effective amount of breast cancer-targeted nanoparticles and pharmaceutically acceptable carriers and / or adjuvants. The drug for oral administration can be made into common dosage forms such as tablets, pills, powders, granules, capsules, emulsions, syrups, ointments, suppositories, etc.; in this embodiment, no specific limitations are imposed on the carriers and / or adjuvants, and the carriers and / or adjuvants can be adaptively adjusted according to the specific drug dosage form.

[0135] Generally, the "effective amount" of a compound (breast cancer-targeted nanoparticles) refers to the amount sufficient to cause a target biological response. As understood by those of ordinary skill in the art, the effective amount of the compound in this embodiment can be changed according to the following factors: for example, components such as the vehicle in the drug, as well as the age and health status of the subject and the symptoms of triple-negative breast cancer.

[0136] Among them, the effective amount includes a therapeutically effective amount and a prophylactically effective amount.

[0137] Unless otherwise stated, the "therapeutically effective amount" of the compound used in this embodiment is the amount sufficient to provide a benefit during the treatment of triple-negative breast cancer, or the amount that minimizes the improvement or alleviation of one or more symptoms (manifestations) related to the triple-negative breast cancer state. The "prophylactically effective amount" of the compound used in this embodiment is the amount sufficient to prevent the occurrence of triple-negative breast cancer, or the amount sufficient to prevent one or more symptoms related to the occurrence of the triple-negative breast cancer state.

[0138] It can be understood that the drug of this embodiment can also be made into an injection. For example, the breast cancer-targeted nanoparticles can be made into corresponding injections with water for injection, normal saline, and glucose water under a sterile operating environment, and the above-mentioned injections can be prepared by conventional methods.

[0139] Next, specific embodiments will be combined to further elaborate on this application. It should be understood that these embodiments are only used to illustrate / explain this application and not to limit the scope of this application.

[0140] In the following examples, the materials, reagents, and instruments used can be obtained commercially without special instructions.

[0141] Example 1

[0142] This example provides a method for preparing breast cancer-targeted nanoparticles.

[0143] Please refer to Figure 1 the synthetic route diagram, and the method for preparing breast cancer-targeted nanoparticles includes:

[0144] (1) Preparation of Polymer I: Folate Albumin Nanocarrier (BSA-FA).

[0145] Weigh 13.3 mg of FA and add it to 5 mL of DMSO solution. Add 11.6 mg of EDC, 6.9 mg of NHS, and 3.0 mg of DMAP to the above reaction solution, and stir at room temperature in the dark for 12 h for activation. Weigh 40 mg of BSA and add it to 5 mL of water, and adjust the pH to 9.9 with 0.2 M NaOH solution, then dissolve it by ultrasonic treatment. Then, drop the FA activation solution into the BSA solution, and stir at 500 rpm / min in the dark at room temperature for 12 h. Dialyze with ultrapure water (14 KDa) for 72 h, and centrifuge at 12,000 rpm for 10 min to obtain the folate albumin nanocarrier.

[0146] (2) Preparation of Polymer II: pH-Sensitive Polymer (mPEGA-DCA-NHS).

[0147] Dissolve 100.0 mg of mPEG-NH2 (mPEGA) and 80.0 mg of 3,4,5,6-tetrahydrophthalic anhydride (DCA) by stirring in 5 mL of water, and adjust the pH to 8.5 with 0.2 M NaOH solution. After 12 h, centrifuge and filter (4000 rpm, 30 min), with a cut-off value of 1 KDa, to obtain the mPEGA-DCA polymer; freeze-dry the dialyzed mPEGA-DCA polymer; dissolve the freeze-dried mPEGA-DCA polymer in water to make an 80 mg / mL mPEGA-DCA solution;

[0148] Take 150.0 mg of EDC·HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 100.0 mg of NHS and add them to 8 mL of the above mPEGA-DCA solution, and stir at room temperature for 12 h. Then, adjust the pH to 8.0 with 0.2 M NaOH solution, centrifuge and filter (4000 rpm, 20 min), with a cut-off value of 1 KD. The purified polymer is mPEGA-DCA-NHS.

[0149] (3) Preparation of Polymer III: pH-Sensitive Folate-Conjugated Albumin Nanocarrier (BSA-FA@mPEG).

[0150] Take 10.0 mg of mPEGA-DCA-NHS and dissolve it in 5 mL of water (pH 7.4) to obtain an aqueous solution of mPEGA-DCA-NHS; take 20 mg of BSA-FA and dissolve it in 5 mL of water to obtain an aqueous solution of BSA-FA; add the aqueous solution of mPEGA-DCA-NHS to the aqueous solution of BSA-FA, and add 0.2 M NaOH solution to adjust the pH to 7.4, and stir at 400 rpm / min at room temperature for 12 h. Unreacted polymers are removed by washing and centrifugation (12000 rpm, 10 min), and dialysis is carried out to obtain the pH-sensitive folate-conjugated albumin nanocarrier BSA-FA@mPEG.

[0151] (4) Preparation of Baicalin-Loaded pH-Sensitive Folate-Conjugated Albumin Nanoparticles (BA-AN-FA@mPEG).

[0152] Take 30 mg of BSA-FA@mPEG in 3 mL of water, and add 0.2 M NaOH solution to adjust the pH to 8.5, and stir at room temperature for 10 min. Weigh 2.5 mg of BA and dissolve it in 1 mL of absolute ethanol, and add it dropwise to the BSA-FA@mPEG solution, and stir at 500 rpm / min for 30 min. Continuously add 11 mL of ethanol at a rate of 1.5 mL / min, and the solution changes from clear to a clear opalescent solution, and continue to stir for 12 h. Centrifuge at 12000 rpm for 20 min, wash twice with ultrapure water, and filter through a 0.45 μm organic filter membrane to obtain the drug-loaded pH-sensitive folate-conjugated albumin nanoparticles. Rotate evaporation is used to remove the organic solvent, and the obtained nanoparticle solution is pre-frozen at -80 °C and then placed in a freeze dryer for 24 h to obtain the freeze-dried powder.

[0153] Example 2

[0154] Next, the BA-AN-FA@mPEG prepared in Example 1 will be tested or verified.

[0155] Specifically include:

[0156] 2.1, Nuclear Magnetic Resonance Hydrogen Spectroscopy (1H-NMR)

[0157] Weigh 40 mg of the BA-AN-FA@mPEG freeze-dried powder, and dissolve it in deuterated DMSO. Using tetramethylsilane (TMS) as the internal standard, the nuclear magnetic hydrogen spectrum of the sample is measured by a nuclear magnetic resonance spectrometer. The test results are as Figure 2 shown.

[0158] Please refer to Figure 2 , Figure 21H NMR spectra of FA, BSA, FA-BSA, BA, mPEGA-DCA-NHS, and BA-AN-FA@mPEG are shown; among them, the 1H NMR spectrum of FA-BSA has a characteristic peak of BSA at 9.8 ppm. At the same time, FA-BSA contains characteristic peaks of aromatic protons of FA at 6.3 - 8.6 ppm, proving the successful coupling of folic acid and BSA. BA-AN-FA@mPEG contains characteristic peaks of aromatic protons of mPEGA-DCA-NHS at 1.0 - 2.7 ppm, proving the successful coupling of the pH-sensitive polymer. BA-AN-FA@mPEG contains characteristic peaks of aromatic protons of BA at 6.6 - 8.7 ppm, further confirming the presence of BA in BA-AN-FA@mPEG.

[0159] 2.2 Infrared Spectroscopy (FTIR)

[0160] Weigh 10 mg of BA-AN-FA@mPEG lyophilized powder and grind it evenly with dry potassium bromide in a ratio of 1:100, then press it into a sample, and use an infrared spectrometer to scan the characteristic absorption peaks of the sample in the range of 4000 - 500 cm -1 . Characteristic absorption peak scans of other samples (FA, BSA, FA-BSA, BA, mPEGA-DCA-NHS, BSA-FA@mPEG) are carried out according to the same method. The test results are as Figure 3 and Figure 4 shown.

[0161] Please refer to Figure 3 and Figure 4 , Figure 3 and Figure 4 respectively show the infrared spectra of FA, BSA, FA-BSA, BA, mPEGA-DCA-NHS, BSA-FA@mPEG, and BA-AN-FA@mPEG; among them, characteristic absorption peaks of BSA and FA can be found in the FTIR spectrum of FA-BSA. The characteristic absorption peaks of BSA are 3304.31 cm -1 and 2958.56 cm -1 . FA couples with the active amino group of BSA to form an amide bond, and new characteristic peaks appear at 1606.80 cm -1 and 1537.63 cm -1 in FA-BSA, which belong to the amide bond.

[0162] At the same time, 1508 cm -1 is the characteristic absorption of (C=O) of the benzene ring, and at 1273.43 cm -1The peak appearing at [specific location] corresponds to the stretching vibration of the folic acid benzene ring (C-O), which can preliminarily confirm the presence of FA in FA-BSA and the successful conjugation of FA with BSA. BA-AN-FA@mPEG at 1654.71 cm -1 and 1534.27 cm -1 The peak values at [specific location] correspond to the characteristic peaks (C=O) of the BA molecule at 1726.55 cm -1 and 1660.08 cm -1 Moreover, BA-AN-FA@mPEG has the characteristic peak of the phenolic hydroxyl group of BA (3393.30 cm -1 ), proving the presence of BA in BA-AN-FA@mPEG.

[0163] To reduce the non-specific absorption of nanoparticles by non-cancer cells, in this study, a pH-sensitive polymer was grafted onto the surface of the nanoparticles to hide the targeting molecule FA. BA-AN-FA@mPEG at 1104.62 cm -1 (C-O), 1457.52 cm -1 (C=O), 3418.81 cm -1 (NH stretching vibration) all show characteristic peaks, and the results indicate that mPEG-DCA-NHS was successfully grafted onto BSA-FA@mPEG.

[0164] 2.3. Thermogravimetric analysis

[0165] The thermal properties of the freeze-dried powders of BSA, BA, BA-AN, BA-AN-FA, and BA-AN-FA@mPEG (10 mg of the sample weight) were studied using a TG analyzer (PerkinElmer, USA), and TG data were recorded under the conditions of 30 °C - 600 °C and a nitrogen flow rate of 10 °C / min. The test results are as Figure 5 and Figure 6 shown.

[0166] Please refer to Figure 5 and Figure 6 for the thermogravimetric analysis diagrams. Thermogravimetric analysis reflects the thermal decomposition and thermal stability of the samples. At 35 - 120 °C, most of the crystal water is released at this stage. A significant mass loss was observed in the temperature range of 200 - 400 °C, which may be related to dehydroxylation and decarbonization.

[0167] Specifically, combining Figure 5 and Figure 6The test results show that the maximum weight loss rates of BA, BSA, FA-BSA, BA-BSANPs, BSA-FA@mPEG, and BA-AN-FA@mPEG are 45%, 36%, 27%, 35%, 65%, and 26% respectively. Compared with BSA (220 °C), the maximum weight loss temperatures of FA-BSA (340 °C) and BSA-FA@mPEG (350 °C) increase, indicating that the interaction between mPEG, FA, and BSA may change the surface properties of the nanoparticles.

[0168] Meanwhile, the degradation temperatures of BA-BSANPs and BA-AN-FA@mPEG are higher than that of BA (254 °C), and the maximum weight loss rate is lower than that of BA, indicating that in the prepared BA-AN-FA@mPEG, the outermost modified mPEGA can improve the stability of BA, thereby improving the overall thermal stability of the nanoparticles.

[0169] 2.4. Morphological Observation

[0170] Dissolve 10 mg of BA-AN-FA@mPEG lyophilized powder in 1 mL of ultrapure water, dilute it 1:10 to obtain the BA-AN-FA@mPEG aqueous solution, drop it on the copper mesh for electron microscopy sample preparation after coating, stain it with 2% phosphotungstic acid negatively, and dry it in the air. Observe the particle morphology with a transmission electron microscope. The schematic diagram of the appearance morphology of the BA-AN-FA@mPEG aqueous solution is as Figure 7 shown, and the schematic diagram of the transmission electron microscopy morphology is as Figure 8 shown.

[0171] Please refer to Figure 7 the schematic diagram of the appearance morphology and Figure 8 the schematic diagram of the transmission electron microscopy morphology of , the BA-AN-FA@mPEG dispersion has a light yellow opalescence, is a transparent colloidal solution, and has good stability. Observed under a transmission electron microscope, BA-AN-FA@mPEG shows a core-shell spherical shape structure, and it can be clearly seen that the outermost layer is wrapped with mPEGA on the surface of the nanoparticles, and the size is uniform and dispersed.

[0172] 2.5. Zeta Potential and Particle Size Measurement

[0173] Dissolve 10 mg of BA-AN-FA@mPEG lyophilized powder in 1 mL of ultrapure water, and measure the particle size and Zeta potential with a particle size analyzer to investigate the particle size and Zeta potential distribution.

[0174] Please refer to Figure 9 the schematic diagram of the particle size and Figure 10 the schematic diagram of the Zeta potential of , as Figure 9As shown, the particle size of BA-AN-FA@mPEG was (185.8±2.36) nm and the PDI was 0.094±0.06, indicating that the prepared nanoparticles had a uniform particle size, good dispersibility, and good stability. As Figure 10 shown, the Zeta potential of the BA-AN-FA@mPEG aqueous solution was -(32.70±1.27) mV. Nanoparticles with negative potential tended to repel each other and were in a stable state, and could remain stable for weeks or even months without aggregation.

[0175] Among them, when entering the acidic tumor environment, the potential value of BA-AN-FA@mPEG will increase, the nanoparticles will be unstable, and the drug will be released relatively quickly.

[0176] 2.6 Determination of Encapsulation Efficiency (EE) and Drug Loading (DL)

[0177] Take 10 mg of BA-AN-FA@mPEG freeze-dried powder and dissolve it in 1 mL of PBS. In an ultrafiltration tube, centrifuge at 12,000 rpm / min. Pipette an appropriate amount of the supernatant into a 10 mL volumetric flask, add methanol to dilute and make up the volume, filter through a 0.45 μm organic filter membrane, inject 10 μL, and determine the content of free drug by HPLC.

[0178] After removing the supernatant, wash it twice with PBS, place it in a 5 mL centrifuge tube, add 2 mL of trypsin solution, incubate at 37 °C for 4 h, then transfer it to a 10 mL volumetric flask, make up the volume with methanol, and place the resulting suspension in an ultrasonic cleaner; ultrasonicate at the maximum power for 10 min, centrifuge at 12,000 rpm for 5 min, take the supernatant, filter through a 0.45 μm organic filter membrane, inject 10 μL, and according to the experimental conditions: injection volume: 10 μL, flow rate: 1.0 mL / min, column temperature: 25 °C, detection wavelength: 280 nm, mobile phase: methanol: 0.2% phosphoric acid (47:53), chromatographic column: C18 column (250×4.6 mm, 5 μm), determine the total drug content by HPLC. The total drug content is Wt, Wf is the amount of free drug; the total amount of nanoparticles, drug and carrier material is W (carrier + drug), and calculate according to the formula: Encapsulation Efficiency (EE) = (Wt - Wf) / Wt * 100%; Theoretical Drug Loading (DL) = Wt * EE / W (carrier + drug).

[0179] Prepare 3 batches of BA-AN-FA@mPEG nanoparticles and determine their encapsulation efficiency and drug loading. The test results are shown in Table 1 below.

[0180] Table 1: Encapsulation efficiency and drug loading of BA-AN-FA@mPEG (n = 3)

[0181] Sample 1 2 3 Mean±SD Entrapment Efficiency (%) 73.75 73.84 73.88 73.82±0.069 Drug Loading (%) 5.67 5.68 5.68 5.68±0.005

[0182] According to the test results in Table 1, the encapsulation efficiency and drug loading of the nanoparticles after optimizing the preparation process in this example are both relatively stable, indicating that the optimized preparation method has good reproducibility and can be adopted.

[0183] 2.7. Stability investigation

[0184] To evaluate the in vitro stability of the nanoparticles, the method described in the literature (Feng Lizhen, Huang Jinping, Liu Shengjun, et al. Process optimization study on the self-assembled nanoparticle drug delivery system of glycyrrhizic acid, curcumin and hydroxycamptothecin [J]. Journal of Guangxi Medical University, 2024, 41(04): 598-607. DOI: 10.16190 / j.cnki.45-1211 / r.2024.04.017.) was used to evaluate the in vitro stability of the nanoparticles.

[0185] Specifically: 20 mg of BA-AN-FA@mPEG lyophilized powder was resuspended in different media: 15 mL of deionized water and 15 mL of PBS solution (pH 7.4). Taking particle size and encapsulation efficiency as evaluation indicators, the stability of the nanoparticles at 4°C and 25°C for three months (1, 2, 4, 8, 12 weeks) was investigated. The test results are as Figure 11 shown.

[0186] Please refer to Figure 11 the schematic diagram of the encapsulation efficiency stability test results. When BA-AN-FA@mPEG is stored at 4°C, the change trend of the encapsulation efficiency is relatively gentle within 12 weeks. At room temperature, the decrease amplitude of the encapsulation efficiency is obvious, and the encapsulation efficiency decreases significantly at 12 weeks. And the decrease trend of the encapsulation efficiency of BA-AN-FA@mPEG after incubation with the same volume of PBS is greater than that in aqueous solution, which may be because the surface charge of the nanoparticles is destroyed by the ions in the PBS solution, and agglomeration and sedimentation are likely to occur, affecting the stability of the nanoparticles. Therefore, the preferred storage condition for the prepared nanoparticles is 4°C aqueous solution.

[0187] 2.8. pH sensitivity evaluation of BA-AN-FA@mPEG

[0188] To study the PEG dissociation of nanoparticles under weakly acidic conditions, BA-AN-FA@mPEG was incubated in PBS at pH 7.4, pH 6.5, and pH 5.5, respectively. Specifically, 13.0 mL of a 10 mg / mL BA-AN-FA@mPEG solution at pH 7.4, 6.5, or 5.5 was incubated at 37 °C. At predetermined time intervals, 0.5 mL of the sample solution was taken out, and 1.0 mL of PBS (pH = 7.4) was added for HPLC analysis according to the conditions of Experiment 2.6 to determine the characteristics of BA-AN-FA@mPEG hydrolysis at pH 7.4, 6.5, or pH 5.5, and the dissociation percentage of the PEG chain was calculated. The particle size and potential changes of BA-AN-FA@mPEG incubated at different pH values for different times were determined. The test results are shown as Figure 12 - Figure 13 follows.

[0189] Please refer to Figure 12 the schematic diagram of the hydrolysis rate test results, in which BA-AN-FA@mPEG was incubated for 48 h under different pH conditions to analyze the hydrolysis of the PEG chain.

[0190] At different time points, the hydrolysis rates of the PEG chains at pH 7.4 or pH 5.5 were similar, reaching 55.34% and 57.43% respectively; the hydrolysis rate of the PEG chain was the highest under incubation in PBS at pH 6.5 and increased significantly with the increase of the incubation time, reaching 88.16% after 48 h. It shows that the hydrolysis range of the PEG chain is around 6.5 and can be selectively decomposed in the acidic tumor microenvironment (pH 6.5 - 6.8).

[0191] Please continue to refer to Figure 13 the schematic diagram of the particle size and potential changes, in which the same batch of prepared nanoparticles was measured to have different zeta potentials at different pH values. The pH dependence of the surface charge can be attributed to the protonation and deprotonation of the amino part of the nanoparticle PEG. In a neutral medium (pH 7.4), the amino group is deprotonated, and anions (HPO4 2- , H2PO 4- or Cl - ) in the medium are adsorbed on the surface of the nanoparticles, resulting in a large surface negative charge.

[0192] As the medium becomes acidic (pH 6.5, 5.5), the amino groups of the nanoparticles are protonated, and the shielding layer of the PEG layer will detach from the surface of the nanoparticles, thus changing the zeta potential. Incubated at different pH values (pH 7.4, 6.5, and 5.5), the particle size and zeta potential of BA-AN-FA@mPEG gradually increase, but still maintain a weak negative potential, which are -25.9 mV, -21.0 mV, and -15.6 mV respectively. Among them, the trends of particle size and potential change of the nanoparticles at pH 6.5 are relatively small. In summary, it shows that the prepared nanoparticles can maintain relative stability at pH 6.5.

[0193] 2.9, Investigation on pH-Sensitive Release of BA-AN-FA@mPEG

[0194] Take 10 mg / mL BA and 10 mg / mL BA-AN-FA@mPEG, and incubate them in PBS (13.0 mL) at pH 7.4, pH 6.5, and pH 5.5 respectively, and release them at a constant speed with shaking in a 37 °C air bath. At the determined time points, 0.5 mL of the external solution is taken out, and the same volume of fresh solution is added to the dialysis solution (MWCO: 14,000 Da), and the in vitro release rate is obtained according to the drug loading conversion.

[0195] Please refer to Figure 14 , Figure 14 shows the schematic diagram of the drug release curves of BA-AN-FA@mPEG and BA. After incubating at pH 6.5 for 48 h, the release rate is approximately 82.0%. At pH 5.5 and 7.4, the release rates are 71.8% and 72.6% respectively, indicating that the pH-sensitive nanoparticles can selectively release drugs under different pH conditions. The results show that BA-AN-FA@mPEG releases drugs faster in an environment of pH 6.5, and the release amount increases with the prolongation of the incubation time, while the release is slower at pH 7.4 and 5.5.

[0196] Specifically, the drug release curves of the nanoparticles under the three pH conditions also prove that there is no burst release behavior. BA is a weakly acidic compound and is relatively stable under acidic conditions. The complete release level can be reached earlier under the condition of pH 7.4 solution. Compared with monomer BA, the drug release rate of BA-AN-FA@mPEG is slower, proving that the nanocarrier has a function of sustained drug release.

[0197] Example 3

[0198] Study on the inhibitory effect and mechanism of the breast cancer-targeted nanoparticles (BA-AN-FA@mPEG) prepared in Example 1 on breast cancer growth.

[0199] (1) Cellular uptake

[0200] To investigate the uptake of BA-AN-FA@mPEG by MDA-MB-231 cells, coumarin nanoparticles were prepared by replacing BA with coumarin-6 (C6), and the final concentration of C-6 was 10 μg / mL.

[0201] Take 5×10 4 MDA-MB-231 cells in logarithmic growth phase were seeded in confocal dishes and cultured for 24 h. 200 ng / mL free C-6, 200 ng / mL BA-AN-FA loaded with C-6, and 200 ng / mL BA-AN-FA@mPEG loaded with C-6 were added respectively, and a blank control group was set. They were incubated in DMEM complete medium at pH 7.4 or 6.5 at 37 °C for 2 h. After washing with PBS, 500 μL of 4% paraformaldehyde was added and fixed for 10 min; washed with PBS, 500 μL of DAPI was added and stained for 10 min, and then left to stand and wash with PBS. 500 μL of PBS buffer was added, and cell uptake was observed under a laser confocal microscope.

[0202] For quantitative analysis of cell uptake, MDA-MB-231 cells were seeded in 6-well plates at a density of 2×10 5 cells / well and cultured in a 5% CO2 incubator at 37 °C for 24 h. Then the cells were treated with drug-containing medium for 2 h, and a blank control group was set. The cells were digested with trypsin without EDTA, centrifuged and collected. The fluorescent dye on the cell surface was removed by washing with PBS. After centrifugation, the cells were resuspended in 500 μL of PBS, transferred to a flow tube, and the fluorescence intensity inside the cells was measured by flow cytometry.

[0203] Please refer to Figure 15 - Figure 17 for Figure 15 the qualitative uptake test results of BA, BA-AN-FA, and BA-AN-FA@MPEG by MDA-MB-231 cells at pH 7.4; Figure 16 the qualitative uptake test results of BA, BA-AN-FA, and BA-AN-FA@MPEG by MDA-MB-231 cells at pH 6.5; Figure 17 the quantitative uptake test results of C-6, BA-AN-FA, and BA-AN-FA@MPEG by MDA-MB-231 cells. Figure 17 In

[0204] Combined with Figure 15 - Figure 17 the test results, compared with the control group, BA-AN-FA@mPEG showed stronger fluorescence signals in cells than BA-AN-FA and C-6, indicating that BA-AN-FA@mPEG can promote BA to penetrate the cell barrier and improve cell-targeted uptake.

[0205] Meanwhile, the fluorescence intensity of BA-AN-FA@mPEG nanoparticles taken up by breast cancer cells at pH 6.5 was stronger than that at pH 7.4. This result indicates that the uptake of BA-AN-FA@mPEG nanoparticles by cells can be promoted at pH 6.5 (under weakly acidic microenvironment conditions).

[0206] (2) Cell viability

[0207] Take the mother liquors of BA, BA-AN-FA, BSA-FA@mPEG, and BA-AN-FA@mPEG with a concentration of 200.0 μg / mL, filter them through a 0.22 μm filter membrane for later use; take MDA-MB-231 cells in the logarithmic growth phase, and dilute the cell suspension to a cell density of 4×10 4 cells / mL. Disinfect a 96-well plate with ultraviolet light for 30 min, add 100 μL of the cell suspension to each well, and place it in an incubator at 37 °C with 5% CO2. After 24 h, take out the 96-well plate, remove the old culture medium, and add 100 μL / well of the drug-containing culture media with different concentrations: BA (1.6, 3.1, 6.3, 12.5, 25.0, 50.0, 100.0, 200.0 μg / mL), AN-FA@mPEG (1.6, 3.1, 6.3, 12.5, 25.0, 50.0, 100.0, 200.0 μg / mL), BA-AN-FA@mPEG (calculated as BA, 1.6, 3.1, 6.3, 12.5, 25.0, 50.0, 100.0, 200.0 μg / mL). Set up 7 concentration gradient groups and a blank control group for the materials, and place the different groups of 96-well plates in the incubator. After a fixed time, take out the 96-well plates, add 10 μL of MTT solution, and place it in an incubator at 37 °C with 5% CO2. After 4 h, take out the 96-well plates, remove the MTT solution and the culture medium, add 150 μL of DMSO solution to dissolve the formazan, gently shake to dissolve the crystals, incubate at 37 °C for 20 min, and record the OD value at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Using the blank group as a control, calculate the cytotoxicity and IC50.

[0208] Please refer to Figure 18 , Figure 18 which respectively show the results of the effects of BA, BA-AN-FA@mPEG, and AN-FA@mPEG on the viability of MDA-MB-231 cells. When the concentration of BA-AN-FA@mPEG was 50.0 μg / mL, it had obvious cytotoxicity, and the cell survival rate was only 46.14%; at the same concentration, when the cells were incubated with monomer BA, the cell survival rate exceeded 60%. Compared with monomer BA, BA-AN-FA@mPEG can significantly reduce cell viability.

[0209] Among them, the IC50 of BA is 63.40 μg / mL, and the IC50 of BA-AN-FA@mPEG is 26.34 μg / mL. Moreover, AN-FA@mPEG has no obvious toxicity to MDA-MB-231 cells in the range of 1.6 - 200.0 μg / mL, and the cell viability is greater than 80%. Therefore, the nanocarrier can promote cell uptake of drugs, thereby enhancing cytotoxicity.

[0210] (3) Cell cycle

[0211] Seed MDA-MB-231 cells at 2×10 5 cells / well in a 6-well plate, culture in a 5% CO2, 37°C incubator, remove the medium, add BA, BA-AN-FA, BSA-FA@mPEG, different concentrations of BA-AN-FA@MPEG (15, 30, 60 μg / mL in terms of BA) and treat for 24 h. After culturing, aspirate the supernatant of the medium and reserve it for later use. Then, digest the adherent cells with trypsin without EDTA, collect the cells into a centrifuge tube, and also add the previously collected medium supernatant into it. Centrifuge at 1000 rpm for 5 min, wash the cells with PBS and centrifuge; resuspend the cells with 0.5 ml PBS, transfer them to a 1.5 ml EP tube, slowly add 1.2 ml of pre-cooled absolute ethanol to make the final concentration of ethanol 75%, and fix overnight at -20°C. After the cells are fixed, centrifuge at 1500 rpm for 5 min, wash the ethanol away with PBS by centrifugation, add 500 μL of cell buffer, and stain with 5 μL of PI / RNAse stain in the dark at room temperature for 15 min. Detect with a flow cytometer. The test results are as Figure 19 - Figure 21 shown.

[0212] In this example, the cell cycle was detected by PI-labeled DNA flow cytometry, Figure 19 - Figure 21 showing the cell cycle test chart of MDA-MB-231. Figure 19 showing the cell cycle test chart of MDA-MB-231 cells after adding different concentrations of BA-AN-FA@MPEG; Figure 20 showing the cell cycle test chart of MDA-MB-231 cells in the blank group and after adding BA, BA-AN-FA, AN-FA@MPEG; Figure 21 showing the cell cycle test chart of MDA-MB-231 cells in the blank group and after adding BA, BA-AN-FA, AN-FA@MPEG, different concentrations of BA-AN-FA@MPEG at different pH values. Figure 19 - Figure 21 Among them, the left side shows the test results at pH 6.5, and the left side shows the test results at pH 7.4.

[0213] According to the test results, compared with the control group, after treatment with different concentration gradients of BA-AN-FA@mPEG (15, 30, 60 μg / mL), the proportion of cells in the G2 / M phase decreased, and the proportion of cells in the S phase increased accordingly.

[0214] Moreover, compared with incubation under pH 7.4 conditions, BA-AN-FA@mPEG could significantly inhibit the entry of MDA-MB-231 cell cycle into the G2 / M phase at pH 6.5, and the degree of change was positively correlated with the dose, indicating that BA-AN-FA@mPEG could significantly induce cell cycle arrest of MDA-MB-231 cells in the S phase under pH 6.5 conditions.

[0215] (4) Cell apoptosis

[0216] Inoculate MDA-MB-231 cells at 2×10 5 cells / well into a 6-well plate, culture them in an incubator with 5% CO2 at 37°C, remove the culture medium, and incubate with fresh culture solutions containing BA, BA-AN-FA, BSA-FA@mPEG, and different concentrations of BA-AN-FA@MPEG (15, 30, 60 μg / mL in terms of BA); after 24 h, collect the supernatant (containing apoptotic suspended cells) for later use. Digest the cells with trypsin without EDTA, mix the previously collected supernatant with the digested cell suspension, centrifuge at 1000 rpm for 5 min, and collect the cell precipitate. Wash the cells with cold PBS (centrifuge at 1000 rpm for 5 min). Resuspend the cells with 500 μL Binding Buffer, transfer them to a flow tube, add 5 μL AnnexinV-APC and 5 μL PI respectively, mix well, incubate in the dark for 15 min, and detect with a flow cytometer. The test results are as Figure 22 - Figure 24 shown.

[0217] Figure 22 The flow cytometry diagrams of cell apoptosis of MDA-MB-231 in the blank control group and after adding BSA-FA@MPEG are shown respectively; Figure 23 The flow cytometry diagrams of cell apoptosis of MDA-MB-231 after adding BA and BA-AN-FA are shown respectively; Figure 24 The flow cytometry diagrams of cell apoptosis of MDA-MB-231 after adding different concentrations of BA-AN-FA@MPEG are shown respectively.

[0218] Combined Figure 22 - Figure 24The test results showed that, at the same concentration, compared with the BA-AN-FA and BA groups, BA-AN-FA@mPEG was more effective in inducing apoptosis in breast cancer MDA-MB-231 cells, indicating that the BA-AN-FA@mPEG nanocarrier could promote the apoptosis-inducing effect of BA. Compared with the pH 7.4 condition (40.1%), the MDA-MB-231 cells treated with BA-AN-FA@mPEG (60 μg / mL) had a higher apoptosis rate (72.9%) at pH 6.5. In addition, the apoptosis of cells treated with the BA-AN-FA and BA groups showed minimal dependence on pH, indicating that BA-AN-FA@mPEG could specifically respond to the weakly acidic conditions of the tumor microenvironment, promote the uptake of breast cancer MDA-MB-231 cells, and effectively induce apoptosis.

[0219] (5) Co-culture system

[0220] Extract exosomes derived from TNBC cells and co-culture them with macrophages, observe the uptake of exosomes by macrophages and detect the phenotypic markers of macrophages by flow cytometry; incubate MDA-MB-231 cells with DMEM containing 10% exosome-free FBS for 2 days, and extract exosomes for culturing macrophages. After 24 h, collect the macrophage supernatant for culturing MDA-MB-231 cells (TNBC). After co-culturing macrophages with TNBC, western blot was used to detect the expression of apoptosis-related pathways in TNBC cells, and to analyze the effect and mechanism of macrophages and BA-AN-FA@mPEG (30 μg / mL) administration on TNBC apoptosis.

[0221] The experimental steps of western blot were as follows:

[0222] Digest cells with trypsin, collect the cell suspension, centrifuge at 2000 rpm for 5 min, wash the cells twice with PBS, and centrifuge at 2000 rpm for 5 min; collect the cell pellet, add 100 μL of cell lysis buffer to each well, and lyse on ice for 30 min; place in a 1.5 mL EP tube, centrifuge at 12000 rpm for 5 min at 4 °C, collect the supernatant and transfer it to a new pre-cooled EP tube. Use a BCA protein assay kit (BCA (Bicinchoninic Acid) protein quantification kit, BCA1-1KT), perform protein quantification according to the instructions, measure the absorbance at 562 nm with an enzyme-linked immunosorbent assay (ELISA) reader, calculate the concentration of each group, and dilute to the same concentration with normal saline; according to protein supernatant:SDS-PAGE protein loading buffer = 4:1, dilute with the loading buffer; incubate in a metal bath at 100 °C for 5 min, cool on ice, centrifuge at 10000 rpm for 5 min at 4 °C, take the supernatant and store it in a -20 °C refrigerator for later use. Perform electrophoresis at 90 V for 30 min and 120 V for 1 h, and stop when the Marker band separates to the bottom edge of the gel. Cut a PVDF membrane and soak it in methanol for 3 min to activate it 10 min before the end of electrophoresis. The sponge and filter paper also need to be soaked in the transfer buffer for 30 min in advance. After the PVDF membrane is activated, transfer it to the transfer buffer; add the transfer buffer to the transfer cassette, stack according to blackboard-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-whiteboard, remove air bubbles, and transfer at 150 mA voltage on ice for 1.5 h. Immerse the PVDF membrane in 5% skim milk blocking solution, and block it slowly with shaking at 90 r / min at room temperature for 2 h; wash 5 times with PBST, 6 min each time; place it in the primary antibody (1:1000, v / v) solution, and incubate on a shaker at 4 °C for 12 h, using β-actin as the protein loading control. Recover the primary antibody solution, wash 5 times with PBST, 6 min each time; place it in the secondary antibody solution of IgG H&L (1:5000, v / v), and shake at 90 r / min at room temperature for 2 h; recover the secondary antibody solution, wash 5 times with PBST, 6 min each time; prepare the ECL chromogenic solution in the dark, incubate the target band for 3 min, and image it with a Tanon-5200 chemiluminescent gel imaging system, and quantitatively analyze it using Image J software. The test results are as Figure 25 - 30 shown.

[0223] To evaluate the effect of exosomes on macrophages, exosomes were collected from TNBC cells to culture M0 macrophages. Exosomes were stained with PKH26 and Hoechst, and the uptake of exosomes by macrophages was detected by fluorescence. Please refer to Figure 25 , and the results showed that a large number of exosomes entered macrophages after 2 h of treatment.

[0224] According to Figure 26 - Figure 28It is shown that macrophages exposed to TNBC cell exosomes exhibit elevated M2 markers; further, in this example, BA-AN-FA@mPEG (30 μg / mL) was added to the above TNBC cells. According to Figure 29 - Figure 30 It is shown that Figure 29 - Figure 30 Protein expression and quantification after administration are shown. Among them, after administration of BA-AN-FA@mPEG (30 μg / mL), a decrease in M2 markers was observed.

[0225] Combined with the above test results, it can be obtained that TNBC cell exosomes can promote the polarization of M0 macrophages into M2, and the effect of TNBC cell exosomes can be inhibited after administration (BA-AN-FA@mPEG).

[0226] In summary, the breast cancer-targeted nanoparticles provided in this example combine the passive targeting of nanoplatforms, the tumor microenvironment-stimuli response, and the active targeting strategy of folic acid, and utilize the EPR effect, PEG detachment, charge inversion, folic acid ligand-receptor binding, etc. to achieve precise targeting and specific response of tumors, comprehensively enhancing the targeted accumulation of drugs in the tumor microenvironment. The breast cancer-targeted nanoparticles have multiple advantages such as high targeting, controlled drug release, increased solubility and absorption rate of poorly soluble drugs, etc., thus being able to enhance the efficacy of drugs and reduce side effects, showing broad application prospects in the field of anti-tumor therapy.

[0227] The technical solutions provided in the embodiments of the present application have been introduced in detail above. Specific examples are used in this article to elaborate on the principles and implementation manners of the embodiments of the present application. The descriptions of the above embodiments are only applicable to help understand the principles of the embodiments of the present application; at the same time, for those of ordinary skill in the art, based on the embodiments of the present application, changes will occur in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present application.

Claims

1. A method for preparing breast cancer targeting nanoparticles, characterized in that: The preparation method comprises: Dissolving folic acid in an organic solution, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and a catalyst, and reacting to obtain a folic acid activation solution; adding the folic acid activation solution to an albumin solution, and reacting to obtain a polymer I; Dissolving methoxypolyethylene glycol-amino and 3,4,5,6-tetrahydrophthalic anhydride in water, adjusting the pH value to 8.0-8.5, and reacting to obtain an mPEGA-DCA solution; adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the mPEGA-DCA solution, adjusting the pH value to 7.4-8.0, and reacting to obtain polymer II; After mixing the aqueous solution of polymer II with the aqueous solution of polymer I, adjusting the pH value to 7.4-8.0, reacting to obtain polymer III; The polymer III is dissolved in water, and the pH value is adjusted to 8.0-8.5 to obtain a polymer III solution; BA is dissolved in ethanol and added to the polymer III solution, and ethanol is added during stirring to obtain an opalescent solution; the opalescent solution is stirred, centrifuged, and filtered to obtain breast cancer targeting nanoparticles.

2. The preparation method according to claim 1, characterized in that: The organic solvent is dimethyl sulfoxide.

3. The preparation method according to claim 1, characterized in that: The catalyst is 4-dimethylaminopyridine.

4. The preparation method according to claim 1, characterized in that: The method of dissolving BA in ethanol and adding the solution to the polymer III, and adding ethanol during stirring to obtain an opalescent solution comprises: BA was dissolved in ethanol and added to the polymer III solution. Ethanol was added at a rate of 1.0-2.0 mL / min during stirring to obtain an opalescent solution.

5. The preparation method according to claim 1, characterized in that: The method for preparing the albumin solution comprises: dissolving albumin in water to obtain a mixed solution; An alkali reagent is added to the mixed solution, and the pH value of the mixed solution is adjusted to 8-10 to obtain an albumin solution.

6. The preparation method according to claim 5, characterized in that: The albumin is at least one of human serum albumin, bovine serum albumin, and recombinant human serum albumin.

7. A breast cancer targeting nanoparticle, characterized in that: The breast cancer targeting nanoparticles are prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the breast cancer targeting nanoparticles according to claim 7 in the preparation of a drug for preventing, alleviating or treating breast cancer.

9. The use according to claim 8, characterized in that The drug is a drug used to promote the active uptake ability of breast cancer cells.

10. The use according to claim 8, characterized in that The drug is an injectable solution or a drug for oral administration.

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